不同基因型甘蔗与慢生根瘤菌BSA6的联合固氮作用

Rhizobia are able to fix N2 as endophytic diazotrophs in association with non-legume plants, such as some sugarcane genotypes. We isolated a Bradyrhizobium sp. strain BSA6, which is able to fix N2 in free-living states and promote sugarcane growth, from wild Erianthus arundinaceum plants. We plan to study the associative nitrogen fixation between strain BSA6 and Guangxi main sugarcane cultivar ROC22 and Brazilian high nitrogen-fixing cultivar RB867515. We will introduce strain BSA6 into sugarcane plants by culturing micropropagated sugarcane plantlets with strain BSA6 and immersing sugarcane germ stems in BSA6 suspensions. We will determine sugarcane N2-fixation in pots and field plots by using 15N isotope dilution method and 15N natural abundance method, respectively. We will label strain BSA6 with constitutively expressed gfp or gfp regulated by nifH promoter and use confocal microscopy, transmission electron microscopy, and immune-gold labeling of GFP to determine patterns of bacterial localization, organization and nif gene expression in sugarcane plants. We will amplify bacterial nifH transcripts from sugarcane plants by RT-PCR and analyze the nifH transcriptoms by denaturing gradient gel electrophoresis, sequencing and bioinformatics to know the active N2-fixing communities and their interactions. Together, this study will show the principal features of the sugarcane-Bradyrhizobium N2-fixation and interaction, show the effects of sugarcane genotypes, planting patterns, and inoculation methods on the sugarcane-Bradyrhizobium N2-fixation, and provide primary information for studying the associative nitrogen fixation between non-legumes and rhizobia and using an efficient sugarcane-rhizobia N2-fixing system in fields.

根瘤菌能以不结瘤方式作为内生固氮菌与甘蔗等非豆科植物联合固氮。我们从野生斑茅体内分离到能在自由生长状态下固氮并促进甘蔗生长的慢生根瘤菌BSA6。本项目研究BSA6与广西主栽甘蔗品种新台糖22号和巴西高固氮品种RB867515的联合固氮作用。用BSA6以不同方式接种甘蔗组培种苗和种茎，用15N同位素稀释法和15N自然丰度法分别检测盆栽和田间甘蔗的联合固氮水平；用组成表达gfp和nifH-gfp标记BSA6，用共聚焦显微、电镜结合免疫金标记GFP检测BSA6在甘蔗体内的定殖组织模式和固氮功能表达模式，用RT-PCR扩增甘蔗体内固氮菌的nifH转录组，用DGGE和测序解析甘蔗体内功能固氮菌的组成和功能，揭示甘蔗与根瘤菌联合固氮的基本特征，了解甘蔗基因型、种植方式和接种方式对甘蔗与根瘤菌联合固氮的作用，为新兴的非豆科作物与根瘤菌联合固氮研究和在田间实现甘蔗与根瘤菌高效联合固氮提供基础信息。

甘蔗是世界上最重要的糖料和能源作物。根瘤菌能以不结瘤方式作为内生固氮菌与甘蔗等非豆科植物联合固氮。本项目研究了慢生根瘤菌BSA6与广西主栽甘蔗品种新台糖22号和巴西高固氮品种RB928064的联合固氮作用。将GFP标记的BSA6菌株与ROC22和RB928064组培苗在1/10 MS培养液中共培养，用激光扫描共聚焦显微镜观察到GFP标记的BSA6细胞定殖在根冠、根毛和根皮层细胞， BSA6侵入RB928064组培苗根的能力较强。接种BSA6能显著促进甘蔗种茎苗和组培苗的生长，ROC22接种BSA6的种茎苗相比对照，干重和氮含量分别提高为11.01%和12.78%，RB928064接种BSA6的种茎苗相比对照，干重和氮含量分别提高为12.4%和18.13%。15N同位素稀释技术检测到接种BSA6对ROC22和 RB928064种茎苗的固氮效率分别为10.41％和12.74％。ROC22接种BSA6的组培苗相比对照，干重和氮含量分别提高为4.5%和7.9%，RB928064接种BSA6的组培苗相比对照，干重和氮含量分别提高为12.2%和25.6%。15N同位素稀释技术检测到接种BSA6对ROC22和 RB928064组培苗的固氮效率分别为8.29％和12.36％。B9和ROC22甘蔗根系的优势细菌分别归于伯克霍尔德菌Burkholderia，根瘤菌属Rhizobium，链霉菌属Streptomyces，慢生根瘤菌属Bradyrhizobium，Pseudonocardiaceae等属的细菌。B9和ROC22甘蔗根系的优势固氮细菌分别归于慢生根瘤菌属Bradyrhizobium，克雷博氏菌属Klebsiella，伯克霍尔德菌Burkholderia，草螺菌属Herbaspirillum等属的细菌。本研究证明了慢生根瘤菌BSA6能与甘蔗联合固氮，为在田间实现甘蔗与根瘤菌高效联合固氮提供基础信息。